The pituitary glycoprotein hormone, follicle stimulating hormone (FSH), is a heterodimer comprised of two non-covalently bound subunits, α and β (Pierce et al, 1981). The α-subunit is interchangeable among the hormones of this family, which include luteinizing hormone (LH), thyrotropin stimulating hormone (TSH) and chorionic gonadotropin (CG), in addition to FSH. The β-subunit, on the other hand, is unique to each hormone and is primarily responsible for the biological specificity of hormone action (see FIGS. 17 and 18 for the sequences of the hFSH α and β subunits, respectively).
Human FSH (hFSH) contains four N-linked carbohydrate moieties, two on each of the α- and β-subunits. A schematic of the carbohydrate moieties on hLH and hFSH is shown in FIG. 15. While the functional significance of these moieties is poorly understood, they are likely to be important for proper protein folding, subunit assembly and secretion of the hormone (Suganuma et al 1989; Feng et al, 1995). The carbohydrate moieties may also be obligatory for signal transduction, although partially deglycosylated hormones show preserved receptor binding (Calvo et al, 1986; Sairam et al, 1982).
Among the glycoprotein hormones, hCG is known to have the longest circulating half-life. This has been attributed to the presence of four O-linked glycosylation sites on the carboxyterminal peptide (CTP) sequence of the β-subunit, corresponding to amino acids 113–145 (Matzuk et al, 1990). In contrast with N-linked sugars, deglycosylation of O-linked moieties does not affect signal transduction, and hCG devoid of this extension maintains its in vitro bioactivity. Schematic examples of N-linked and O-linked carbohydrates are shown in FIG. 16.
Instead, the importance of the O-linked sugars lies in providing enhanced stability of the hormone in vivo. This was initially deduced from comparisons between hCG and hLH, whose biological activity and β subunits are remarkably similar but whose serum half lives are dramatically different. The β subunits of hCG and hLH share greater than 85% sequence identity through the N-terminal 113 amino acids (Pierce et al, 1981). In addition, these two hormones share a common receptor and elicit similar biologic activity following receptor binding. However, the serum half-life of hCG is almost five-times that of hLH (Porchet et al, 1995; Saal et al, 1991; Yen et al, 1968). The primary structural difference between β-hCG and β-hLH is the additional carboxy-terminal amino acids comprising the CTP sequence of β-hCG. This carboxy-terminal peptide, specifically its O-linked glycosylation sequences, is thus likely to be responsible for both the decreased metabolism and excretion of hCG, and thus also for its notably increased serum half-life over the relatively transient hLH.
The importance of the CTP in promoting hormone stability was demonstrated by the construction of a fusion protein consisting of the CTP portion of the β-subunit of hCG and the carboxy terminus of β-hFSH. This β-hFSH-CTP fusion produced a long-acting hFSH agonist which was able to dimerize with a coexpressed α-subunit to produce a functional FSH hormone (Fares et al, 1992). Importantly, this β-hFSH-CTP demonstrated similar in vitro bioactivity and substantially increased in vivo bioactivity compared with preparations of native hFSH.
Thus, merely adding the CTP sequence to β-hFSH was sufficient to increase the biological activity of the hormone, most likely through an increase in serum-half life. Indeed, recent pharmacokinetic parameter estimates in humans have demonstrated that this β-hFSH-CTP analog has an elimination half-life of 2 to 3 times longer than that of native recombinant hFSH (Bouloux et al, 2001).
Current pharmacologic formulations of FSH include purified urinary derivatives and, more recently, recombinant human FSH (r-hFSH). Due to its relatively short half-life, hFSH must be administered as a daily intramuscular or subcutaneous injection, often for 8 to 12 days when used for ovulation induction (LeContonnec et al, 1994). These regimens of controlled ovarian hyperstimulation are associated with a number of side effects, including local irritation and discomfort, which result in poor compliance and a reduction in therapeutic efficacy.
A long-acting FSH formulation requiring less frequent administration would provide an important development for subjects requiring gonadotropin replacement therapy.
The present invention is based on the surprising and unexpected finding that the addition of multiple N-linked glycosylation sequences confers increased protein stability, as demonstrated by an increased in vivo serum half-life. Importantly, the addition of these novel N-linked moieties does not alter the biological activity of the hFSH analogues disclosed herein, as might have been expected given the known importance of N-linked sugars in signal transduction initiated by gonadotropin hormones. Thus, the present invention provides novel hFSH analogues that have an increased serum half-life without sacrificing biological activity, offering a significant advantage over current technologies in gonadotropin replacement therapy. Such therapies are important for the treatment of infertility and have particular relevance to increasing the efficacy of in vitro fertilization protocols, both in agriculturally important mammals and in humans.